Vaccines comprising acapsular P. multocida hyaE deletion mutants

ABSTRACT

Acapsular hyaE deletion mutants of  P. multocida  can be administered to mammals, particularly ungulates, or birds to provide protective immunity against wild-type  P. multocida,  e.g., to prevent or reduce the severity of hemorrhagic septicemia or pneumonia in mammals, particularly livestock, ungulates, and companion animals, or fowl cholera in birds, particularly poultry.

This application claims the benefit of and incorporates by reference tonow abandoned provisional application Ser. No. 60/608,931 filed Jul. 2,2003.

FIELD OF THE INVENTION

The invention relates to acapsular mutants of Pasteurella multocida andvaccines comprising the mutants.

BACKGROUND OF THE INVENTION

Pasteurella multocida (P. multocida) is associated with a variety ofdiseases, including calf and yearling meningoencephalitis, lamblymphadenitis, horse and donkey septicemia, bovine septicemicpasteurellosis (hemorrhagic septicemia, barbone), swine pasteurellosis,porcine septicemic pasteurellosis, pneumonia, and fowl cholera.

There is a need in the art for effective vaccines that can be used toprovide protective immunity against diseases caused by P. multocida.

SUMMARY OF THE INVENTION

One embodiment of the invention is an isolated Pasteurella multocida (P.multocida) bacterium of serogroup A which comprises a deletion of all ora part of a hyaE gene. The deletion attenuates the bacterium.

Another embodiment of the invention is a vaccine for inducing protectiveimmunity against wild-type P. multocida. The vaccine comprises P.multocida bacterium of serogroup A which comprises a deletion of all ora part of a hyaE gene and a pharmaceutically acceptable vehicle. Thedeletion mutation attenuates the bacterium.

Yet another embodiment of the invention are feeds suitable for mammals(including livestock, ungulates, and companion animals or birds(preferably poultry) comprising a P. multocida bacterium of serogroup Awhich comprises a deletion of all or a part of a hyaE gene. The mutationattenuates the bacterium.

Even another embodiment of the invention is a method of inducingprotective immunity against wild-type P. multocida. A P. multocidabacterium of serogroup A is administered to an animal subject such as amammal (including livestock, ungulates, and companion animals) or a bird(including poultry). The bacterium comprises a deletion of all or a partof a hyaE gene, which attenuates the bacterium. The bacterium therebyconfers to the animal subject protective immunity against wild-type P.multocida.

The invention thus provides tools and methods for inducing protectiveimmunity against diseases caused by P. multocida.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1. Illustration of the construction of hyaE deletion mutants. FIG.1A, schematic showing planned deletion. FIG. 1B, plasmid containingdeleted hyaE insert.

DETAILED DESCRIPTION OF THE INVENTION

Acapsular hyaE deletion mutants of P. multocida serogroup A can beadministered to mammals (including livestock, ungulates, and companionanimals) and birds (including poultry) to provide protective immunityagainst wild-type P. multocida, e.g., to prevent or reduce the severityof diseases such as hemorrhagic septicemia or pneumonia in livestock,ungulates, and companion animals and to prevent or reduce the severityof fowl cholera in birds, especially poultry, respectively. The terms“acapsular mutant(s),” “acapsular bacterium(a),” and “mutantbacterium(a)” are used interchangeably in this description.

Acapsular hyaE Deletion Mutants of P. Multocida Serogroup A

Acapsular hyaE deletion mutants of serogroup A P. multocida (e.g.,serotypes A:1, A:3, A:4) can be generated by mutagenizing the hyaE genein the capsule biosynthetic locus. The genes of the capsule biosyntheticlocus in serogroup A are well known and have been completely sequenced.Chung et al., FEMS Microbiol. Lett. 166(2), 289-96, 1998. The serotypeA:1 locus contains four open-reading frames (ORFs) in region 1 (hexA,hexB, hexC, and hexD), five ORFs in region 2 (hyaA, hyaB, hyaC, hyaD,and hyaE), and two ORFs in region 3 (phyA and phyB).

In certain embodiments, the mutant bacteria do not comprise anyantibiotic resistance genes or foreign DNA, which improves theirenvironmental and ecological attractiveness. A method of generatingdeletion mutants is described in Example 1, but any other methods knownin the art can be used to generate deletion mutations. Simple tests forconfirming that mutants have an acapsular phenotype also are disclosedin Example 1.

P. multocida bacteria with any deletion of all or a part of the hyaEgene is within the scope of the invention so long as the deletionattenuates the bacterium. In one embodiment, the deletion mutationresults in the deletion of amino acids 239-359 of the hyaE protein(i.e., none of amino acids 239-359 are present in the encoded hyaEprotein). In other embodiments, the mutated hyaE open reading framecomprises SEQ ID NO:7 or SEQ ID NO:8.

A mutant bacterium is attenuated if, after exposure to the mutantbacterium, an increase in the dosage of wild-type P. multocida isrequired to kill half the susceptible target species (i.e., the LD₅₀increases) and/or there is a reduction in pathologic lesions (e.g.,pneumonia) after exposure of the target species to the mutant bacteriumcompared with exposure to a wild-type bacterium, and/or there is areduction in commensal colonization of mucosal surfaces where P.multocida reside after exposure of the target species to the mutantbacterium when compared with exposure to a wild-type bacterium.

Attenuation can be assessed by various means as is known in the art. Forexample, for septiceaemic disease (such as fowl cholera), susceptiblespecies can be exposed to wild-type organisms by intranasal,intravenous, intramuscular, or intraperitoneal routes, and the dosagerequired to cause disease between wild-type and mutant organisms can becompared. For pneumonic disease, one can expose susceptible animals towild-type organisms by intranasal, intratracheal, or intrapulmonicroutes and compare the extent and severity of clinical symptoms andpathologic lesions between mutant- and wild-type-exposed animals. Formucosal colonization, animals can be exposed to wild-type organisms byoral, intranasal, or intratonsillar routes and the numbers of organismsrecovered from nasal mucus or tonsillar wash specimens at intervalsafter exposure can be compared.

Vaccine Preparations

Vaccines comprising mutant bacteria can be given alone or as a componentof a polyvalent vaccine, i.e., in combination with other vaccines.Mutant bacteria in a vaccine formulation can be live or killed; eitherlive or killed bacteria can be lyophilized and, optionally,reconstituted as is known in the art. Vaccines can conveniently beprovided in kits, which also can comprise appropriate labeling andinstructions for administering a vaccine to an animal subject (e.g.,livestock, an ungulate, a companion animal) or a bird (e.g., poultry).

Vaccines comprising acapsular mutants also can comprise pharmaceuticallyand veterinarily acceptable carriers. Such carriers are well known tothose in the art and include, but are not limited to, large, slowlymetabolized macromolecules, such as proteins, polysaccharides,polylactic acids, polyglycolic acids, polymeric amino acids, amino acidcopolymers, and inactive virus particles. Pharmaceutically andveterinarily acceptable salts can also be used in the vaccine, forexample, mineral salts such as hydrochlorides, hydrobromides,phosphates, or sulfates, as well as the salts of organic acids such asacetates, proprionates, malonates, or benzoates. Vaccines also cancontain liquids, such as water, saline, glycerol, and ethanol, as wellas substances such as wetting agents, emulsifying agents, or pHbuffering agents. Liposomes also can be used as carriers for mutantbacteria. See U.S. Pat. No. 5,422,120, WO 95/13796, WO 91/14445, or EP524,968 B1.

If desired, an adjuvant can be added to a vaccine. Useful adjuvantsinclude, without limitation, surfactants (e.g., hexadecylamine,octadecylanine, lysolecithin, dimethyldioctadecylammonium bromide,N,N-dioctadecyl-n′-N-bis(2-hydroxyethylpropane di-amine),methoxyhexadecylglycerol, and pluronic polyols); polyanions (e.g.,pyran, dextran sulfate, poly IC, polyacrylicacid, carbopol), peptides(e.g., muramyl dipeptide, dimethylglycine, tuftsin), oil emulsions,alum, and mixtures thereof.

Treatment of Mammals, Particularly Livestock, Ungulates, and CompanionAnimals

“Mammals” include monotremes (e.g., platypus), marsupials (e.g.,kangaroo), and placentals, which include livestock (domestic animalsraised for food, milk, or fiber such as hogs, sheep, cattle, and horses)and companion animals (e.g., dogs, cats). “Ungulates” include, but arenot limited to, cattle (bovine animals), water buffalo, bison, sheep,swine, deer, elephants, and yaks. Each of these includes both adult anddeveloping forms (e.g., calves, piglets, lambs, etc.). Bacteria of theinvention can be administered either to adults or developing mammals,preferably livestock, ungulates, or companion animals.

A convenient method of delivering a bacterium of the invention tomammals (such as livestock, ungulates, or companion animals) is by oraladministration (e.g., in the feed or drinking water or in bait). It isparticularly convenient to top-dress or mix feed with the bacteria.Typically, large animals (e.g., livestock/ungulates such as cattle) aredosed with about 10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸, 5×10⁸, 10⁹, 5×10⁹, or 10¹⁰cfu; about 10⁸, 5×10⁸, 10⁹, 5×10⁹ cfu if feed is top-dressed. Doses ofabout 10⁶ to about 10⁸, about 2×10⁶ to about 3×10⁸, about 2.4×10⁶ toabout 2.6×10⁸, about 10⁴ to about 10⁶ cfu or of about 10⁴ to about 10⁹cfu can be given. Doses can be adjusted for smaller livestock/ungulatessuch as sheep (e.g., about 10⁴, 5×10⁴, 10⁵, 5×10⁵, 10⁶, 5×10⁶, 10⁷,5×10⁷, 10⁸, 5×10⁸ cfu). Analogous dosing regimens can be readily deducedfor companion animals.

Although the oral route is preferred for ease of delivery, other routesfor vaccination can also be used. These include without limitation,subcutaneous, intramuscular, intravenous, intradermal, intranasal,intrabronchial, etc. Bacteria of the invention can be implanted in theear. Bacteria also can be administered by airspray, by eye inoculation,or by scarification.

Treatment of Birds

“Birds” include wild (e.g., game fowl) and domesticated (e.g., poultryor pet) birds and includes both adult and developing forms (e.g.,hatchlings, chicks, poults, etc.). “Poultry” or “poultry birds” includeall birds kept, harvested, or domesticated for meat or eggs, includingchicken, turkey, ostrich, game hen, squab, guinea fowl, pheasant, quail,duck, goose, and emu.

Bacteria of the invention can be administered to a bird by any known orstandard technique, including mucosal or intramuscular injection. In ahatchery, bacteria can be administered using techniques such as in ovovaccination, spray vaccination, or subcutaneous vaccination. On thefarm, bacteria can be administered using techniques such asscarification, spray vaccination, eye drop vaccination, in-watervaccination, in-feed vaccination, wing web vaccination, subcutaneousvaccination, and intramuscular vaccination.

Effective doses depend on the size of the bird. Doses range and canvary, for example, from about 10², 5×10², 10³, 5×10³, 10⁴, 5×10⁴, 10⁵,5×10⁵, 10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸, 5×10⁸, 10⁹, to 5×10⁹ cfu.

All patents and patent applications cited in this disclosure areexpressly incorporated herein by reference. The above disclosuregenerally describes the present invention. A more complete understandingcan be obtained by reference to the following specific examples, whichare provided for purposes of illustration only and are not intended tolimit the scope of the invention.

EXAMPLE 1 Generation of Acapsular hyaE Deletion P. Multocida Mutants

P. multocida mutants of strains 1059 (avian, serotype A:3) and 1062(bovine, serotype A:3) were generated by deleting the coding region oftheir hyaE genes, which blocked synthesis of the capsular building blockN-acetyl-D-glucosamine. The coding regions of the 1059 and 1062 hyaEgenes (SEQ ID NOS:5 and 6, respectively) were obtained by PCRamplification, using the forward primer 5′-ATGAAAAAGGTTAATATCATTGG-3′(SEQ ID NO:1) and the reverse primer 5′-TTAACCTTGCTTGAATCGTTTACC-3′ (SEQID NO:2). All primers were synthesized with an oligonucleotidesynthesizer (Applied Biosystems Inc.) by Integrated DNA Technologies,Inc., Coralville, Iowa. The PCR reactions were carried out using theGeneAmp LX PCR Kit (PE Applied Biosystems, Foster City, Calif.) in aPerkin Elmer GeneAmp 9600 thermocycler. Reaction conditions were 30cycles, with 30 seconds at 95° C., 45 seconds at 48° C., and 60 secondsat 72° C. per cycle.

The two PCR-generated hyaE fragments initiated at their start Met codonsand ended at their stop codons. The PCR fragments were ligated intopCR2.1 (Invitrogen Inc., LaJolla, Calif.) and electroporated into the E.coli strain DH11S (Life Technologies, Rockville, Md.), which generatedthe plasmids pCR2.1hyaE1059 and pCR2.1hyaE1062. The two plasmidconstructs were isolated by the alkaline SDS method, then purified byCsCl centrifugation using standard methods. Both strands of both hyaEgenes were sequenced using the Dye Terminator Chemistry kit from PEApplied Biosystems. Samples were run on an ABI Prism 377 DNA Sequencerby the Nucleic Acids Facility, Iowa State University, Ames, Iowa.

The structural hyaE genes of each strain were 1869 bp in length. Due tosequence variation, primarily found in the 3′ end of the coding region,unique hyaE replacement plasmids were constructed for each strain inorder to engineer the two mutant strains of P. multocida. Precisedeletions within each hyaE gene contained on plasmids pCR2.1hyaE1059 andpCR2.1hyaE1062 were achieved by PCR using the deletion primers5′-AAAGATATCTTGGTTTACTTCAATAATTTC-3′ (SEQ ID NO:3) and5′-AAAGATATCACTGCATCTGTTCAATCAACGAGC-3′ (SEQ ID NO:4). The deletionprimers anneal to the cloned gene approximately 300 base-pairs apart butextend outwards, towards the plasmid vector, rather than inwards. ThePCR reaction amplifies the entire cloning vector and two ends of thegene insert but not the approximately 300 base pair “deletion.” Theamplified product is linear, with the two hyaE gene fragments on theends and plasmid vector in the middle. The primers contain EcoRV sites(GATATC) on their 5′ termini. The ends of the linear amplified product,therefore, contain EcoRV sites. See FIGS. 1A and 1B.

The resulting PCR fragments were subjected to EcoRV restrictiondigestion to remove specific sequences from their ends. Each fragmentwas then ligated to generate inframe deletions excising nucleotides 715through 1082. An eight basepair SmaI linker (5′-CCCCGGGG-3′) wasinserted into the EcoRV deletion site of P. multocida 1062 to ensurethat the mutation would result in an acapsular phenotype. No such DNAwas inserted into the deletion site of the P. multocida 1059 hyaEinsert.

The nucleotide sequences of the 1059 and 1062 mutated hyaE fragments areshown in SEQ ID NOS:7 and 8, respectively. In both 1059 and 1062, aminoacid 239 through amino acid 359 of the hyaE protein were deleted. Inaddition, in both strains the amino acid at former position 360 waschanged from leucine to isoleucine. Strain 1062 received an additional 8amino acids in the deletion site just upstream from the former position360 (Pro Arg Gly Pro Gly Ala Pro Gly; SEQ ID NO:9).

The mutated hyaE fragments were subcloned into EcoRI sites within themultiple cloning site of plasmid pBCSK (Strategene Inc.), followed byinsertion of the Tn903 kanamycin resistance element into the adjacentBamHI sites to produce pBCSKΔhyaEkan^(r)1059 and pBCSKΔhyaEkan^(r)1062,respectively. Construction of the replacement plasmids were completed byligating BssH2 generated ΔhyaEkan^(r) fragments of 1059 and 1062 withthe 1.2 kb temperature-sensitive origin of replication of pBB 192excised from the BssH2 site of pBCSK (Stratagene, Inc.). See U.S. Pat.No. 5,840,556. Because ColE1 origin is inactive in P. multocida, onlythe ligation products generating plasmids p192oriΔhyaEkan^(r)1059 orp192oriΔhyaEkan^(r)1062 were capable of replicating within the hosts.

Replacement plasmids were introduced into the appropriate P. multocidastrains by electroporation. Cells were grown in Columbia broth, thenprepared for electroporation by the following steps. The growth waspelleted by centrifugation at 5000×g for 15 minutes and washed once in100 ml 272 mM sucrose at 0° C. The cell pellet was resuspended (1:3packed bacteria: 272 mM sucrose) on ice. Competent bacteria (100 μl)were mixed in a 0.1 cm electroporation cuvette (Bio-Rad) with 200 ngplasmid DNA which was either unmethylated or methylated in vitro usingHhaI. Immediately after adding DNA, the cells were electroporated (Genepulser, Bio-Rad) at 18,000 V/cm, 800 ohm, and 25 mFd, with resultanttime constants ranging from 11 to 15 msec.

Columbia broth (1 ml, 0° C.) was added to the electroporated cells, andthe resuspensions were incubated at 25° C. for approximately 25 minutes.The cells recovered at 30° C. for 2 hours and were then plated ontoColumbia agar plates containing 50 μg/ml kanamycin. Colonies werevisible after 24-hour incubation at 30° C. Transformed cells were grownat 30° C. overnight in broth containing kanamycin. The cells were thenplated onto dextrose starch agar plates with 50 μg/ml kanamycin andgrown at 3° C. for 24 hours.

Cells possessing integrated plasmid survived antibiotic selection at thenon-permissive temperature for plasmid replication, and these could beidentified because integration of replacement plasmid resulted in anacapsular phenotype. Distinguishing between capsular and acapsular P.multocida colonies of strains 1059 and 1062 grown on dextrose starchagar plates was easily accomplished. Wild-type capsular colonies of thetwo strains possess hyaluronic acid, the major capsule component, whichrenders colonies mucoid in appearance; when viewed under obliquelytransmitted light, these colonies exhibit pearl-like iridescence. Incontrast, the acapsular single-crossover mutants are non-mucoid andnon-iridescent.

Single-crossover mutants were transferred to 5 ml Columbia broth withoutantibiotic supplementation and incubated at 30° C. overnight to resolveplasmid from the chromosome. Growth was transferred to dextrose-starchagar plates without supplemental antibiotic which were incubated at 38°C. for 12 hours. The initial test to identify double crossover mutantsinvolved replica-plating arrays of cells that appeared acapsular ontodextrose starch agar plates, either with or without antibiotic, andgrowing the cells at 38° C. Acapsular colonies that failed to grow onthe antibiotic plates were subjected to PCR analysis using the hyaEforward and reverse primers. The sizes of the PCR products were comparedto those of the wild-type parent using agarose gel electrophoresis.

Suspected P. multocida 1059 and 1062 hyaE deletion mutants were alsoassayed for the presence of the kanamycin resistance gene. Putativemutants possessing “clean” hyaE deletions were devoid of antibioticresistance sequences. A disc diffusion enzyme assay (Kirby-Bauer test,Bauer et al., Am. J. Clin. Path. 45, 493-96, 1966) and an Indian inkstaining assay (Collins & Lyne, MICROBIOLOGICAL METHODS, Butterworths,Boston, Mass., 1976, p. 110) were used to confirm that the two hyaEmutants possessed an acapsular phenotype.

EXAMPLE 2 Vaccination of Turkey Poults with the 1059 ΔhyaE P. MultocidaStrain

Attenuation of the 1059 ΔhyaE P. multocida strain was assessed in threeweek-old broad-breasted white turkey poults. Groups consisting of fourpoults were injected intramuscularly with ten-fold serial dilutions ofexponential growth-phase cultures of wild-type or acapsular mutantcultures suspended in trypticase broth (0.1 ml). The poults wereobserved for 7 days after challenge.

The LD₅₀ values (i.e., the amount of bacteria needed to kill half thepoults) were <10³ organisms for poults injected with the wild-typestrain and in excess of 10⁷ organisms for poults injected with the hyaEmutant.

EXAMPLE 3 Vaccination of Steer Calves with the 1062 ΔhyaE P. MultocidaStrain

Six Holstein steer calves, approximately 500 pounds, were exposed to the1062 ΔhyaE P. multocida acapsular mutant. The strain was administeredeither by subcutaneous injection in the neck (10⁸ cfu) or bytop-dressing feed (10⁹ cfu).

No adverse reaction to either exposure was observed. Mucosal vaccinatesbecame colonized in the palatine tonsils for the remainder of the trial(5 weeks). Intratracheal challenge with the virulent parent strain (10¹⁰cfu total dose in 15 ml) elicited transient (1-2) day fevers in controlcalves but little or no pneumonic changes 10 days post-challenge incontrols or vaccinates.

EXAMPLE 4 Efficacy Study in 2-3 Month-Old Calves ChallengedTranstracheally with Virulent P. Multocida

Three groups of male bovine calves (n=15) were vaccinated subcutaneouslywith a single 2 mL dose of either a 2.6×10⁸ cfu/dose of the 1062 ΔhyaEP. multocida acapsular mutant, a 2.4×10⁶ cfu/dose of the mutant, orsterile PBS. At the time of vaccine administration, the average weightof the calves was 70 kg (154 lbs). After 21 days, calves were challengedtranstracheally with a live culture of P. multocida and observed for 7days.

The mean lung lesion score for the high dose of P. multocida 1062 ΔhyaEwas 3.1±2.8%. The mean lung lesion score for the low dose of P.multocida 1062 ΔhyaE was 3.7±2.2%, and the mean lung lesion score forthe control group was 7.7±5.2%. The median lung lesion score for eachvaccine group was significantly lower than the control group; P<0.0006.

1. An isolated Pasteurellaceae multocida (P. multocida) bacterium ofserogroup A which comprises a hyaE gene comprising a deletion in itscoding region, wherein the deletion results in an acapsular phenotype ofthe bacterium, thereby attenuating the bacterium.
 2. The bacterium ofclaim 1 which comprises no antibiotic resistance genes.
 3. The bacteriumof claim 1 which comprises no exogenous DNA.
 4. The bacterium of claim 1which is serotype A:3.
 5. The bacterium of claim 1 which is serotypeA:1.
 6. The bacterium of claim 1 which is serotype A:4.
 7. The bacteriumof claim 1 wherein the hyaE gene encodes a hyaE protein in which aminoacids 239-359 are deleted, wherein the amino acids are numberedaccording to the hyaE protein encoded by the nucleotide sequence shownin SEQ ID NO:5.
 8. The bacterium of claim 1 wherein the hyaE genecomprising the deletion in its coding region comprises the nucleotidesequence shown in SEQ ID NO:7.
 9. The bacterium of claim 1 wherein thehyaE gene comprising the deletion in its coding region comprises thenucleotide sequence shown in SEQ ID NO:8.
 10. The bacterium of claim 1which is live.
 11. The bacterium of claim 1 which is lyophilized. 12.The bacterium of claim 1 which is killed.
 13. A vaccine for inducingprotective immunity against wild-type P. multocida comprising: thebacterium of claim 1; and a pharmaceutically acceptable vehicle.
 14. Thevaccine of claim 13 wherein the bacterium comprises no antibioticresistance genes.
 15. The vaccine of claim 13 wherein the bacteriumcomprises no exogenous DNA.
 16. The vaccine of claim 13 wherein thebacterium is serotype A:3.
 17. The vaccine of claim 13 wherein thebacterium is serotype A:1.
 18. The vaccine of claim 13 wherein thebacterium is serotype A:4.
 19. The vaccine of claim 13 wherein the hyaEgene comprising the deletion in its coding region comprises thenucleotide sequence shown in SEQ ID NO:7.
 20. The vaccine of claim 13wherein the hyaE gene comprising the deletion in its coding regioncomprises the nucleotide sequence shown in SEQ ID NO:8.
 21. The vaccineof claim 13 wherein the bacterium is live.
 22. The vaccine of claim 13wherein the bacterium is lyophilized.
 23. The vaccine of claim 13wherein the bacterium is killed.
 24. The vaccine of claim 13 which ispackaged with instructions for administering the vaccine to an ungulateto confer protective immunity against wild-type P. multocida.
 25. Thevaccine of claim 13 which is packaged with instructions foradministering the vaccine to a bird to confer protective immunityagainst wild-type P. multocida.
 26. A feed suitable for ungulatescomprising the bacterium of claim
 1. 27. The feed of claim 26 whereinthe bacterium comprises no antibiotic resistance genes.
 28. The feed ofclaim 26 wherein the bacterium comprises no exogenous DNA.
 29. The feedof claim 26 wherein the bacterium is serotype A:3.
 30. The feed of claim26 wherein the bacterium is serotype A:1.
 31. The feed of claim 26wherein the bacterium is serotype A:4.
 32. The feed of claim 26 whereinthe hyaE gene comprising the deletion in its coding region comprises thenucleotide sequence shown in SEQ ID NO:7.
 33. The feed of claim 26wherein the wherein the hyaE gene comprising the deletion in its codingregion comprises the nucleotide sequence shown in SEQ ID NO:8.
 34. Thefeed of claim 26 wherein the bacterium is live.
 35. The feed of claim 26wherein the bacterium is lyophilized.
 36. The feed of claim 26 whereinthe bacterium is killed.
 37. The feed of claim 26 which is packaged withinstructions for administering the feed to ungulates to conferprotective immunity against wild-type P. multocida.
 38. A feed suitablefor a bird comprising the bacterium of claim
 1. 39. The feed of claim 38wherein the bacterium comprises no antibiotic resistance genes.
 40. Thefeed of claim 38 wherein the bacterium comprises no exogenous DNA. 41.The feed of claim 38 wherein the bacterium is serotype A:3.
 42. The feedof claim 38 wherein the bacterium is serotype A:1.
 43. The feed of claim38 wherein the bacterium is serotype A:4.
 44. The feed of claim 38wherein the hyaE gene comprising the deletion in its coding regioncomprises the nucleotide sequence shown in SEQ ID NO:7.
 45. The feed ofclaim 38 wherein the hyaE gene comprising the deletion in its codingregion comprises the nucleotide sequence shown in SEQ ID NO:8.
 46. Thefeed of claim 38 wherein the bacterium is live.
 47. The feed of claim 38wherein the bacterium is lyophilized.
 48. The feed of claim 38 whereinthe bacterium is killed.
 49. The feed of claim 38 which is packaged withinstructions for administering the feed to a bird to confer protectiveimmunity against wild-type P. multocida.
 50. A method of inducingprotective immunity against wild-type P. multocida comprising the stepof: administering the bacterium of claim 1 to an ungulate or a bird,whereby the bacterium confers to the ungulate or bird protectiveimmunity against wild-type P. multocida.
 51. The method of claim 50wherein the bacterium comprises no antibiotic resistance genes.
 52. Themethod of claim 50 wherein the bacterium comprises no exogenous DNA. 53.The method of claim 50 wherein the bacterium is serotype A:3.
 54. Themethod of claim 50 wherein the bacterium is serotype A:1.
 55. The methodof claim 50 wherein the bacterium is serotype A:4.
 56. The method ofclaim 50 wherein the hyaE gene comprising the deletion in its codingregion comprises the nucleotide sequence shown in SEQ ID NO shown in SEQID NO:7.
 57. The method of claim 50 wherein the hyaE gene comprising thedeletion in its coding region comprises the nucleotide sequence shown inSEQ ID NO shown in SEQ ID NO:8.
 58. The method of claim 50 wherein thebacterium is live.
 59. The method of claim 50 wherein the bacterium islyophilized.
 60. The method of claim 50 wherein the bacterium is killed.61. The method of claim 50 wherein the bacterium is administered to anungulate.
 62. The method of claim 61 wherein the ungulate is a bovineanimal.
 63. The method of claim 50 wherein the bacterium is administeredto a bird.
 64. The method of claim 50 wherein the bacterium isadministered to a bird and the bird is a poultry bird.
 65. The method ofclaim 50 wherein the bacterium is administered to a bird and the bird isa poultry bird is selected from the group consisting of chicken, turkey,ostrich, game hen, squab, guinea fowl, pheasant, quail, duck, goose, andemu.
 66. The method of claim 50 wherein the bacterium is administered toa bird and the bird is a chicken.
 67. The method of claim 50 wherein thebacterium is administered to a bird and the bird is a turkey.
 68. Themethod of claim 50 wherein the bacterium is administered subcutaneously.69. The method of claim 50 wherein the bacterium is administeredintramuscularly.
 70. The method of claim 50 wherein the bacterium isadministered orally.
 71. The method of claim 50 wherein the bacterium isadministered by top-dressing feed.
 72. The method of claim 50 whereinthe bacterium is administered intranasally.
 73. The method of claim 50wherein the bacterium is administered at a dose between about 10⁴ andabout 10⁹ cfu.
 74. The method of claim 50 wherein the dose is betweenabout 10⁴ and about 10⁶ cfu.
 75. The vaccine of claim 13, wherein thehyaE gene encodes a hyaE protein in which amino acids 239-359 aredeleted, wherein the amino acids are numbered according to the hyaEprotein encoded by the nucleotide sequence shown in SEQ ID NO:5.
 76. Thefeed of claim 26 wherein the hyaE gene encodes a hyaE protein in whichamino acids 239-359 are deleted, wherein the amino acids are numberedaccording to the hyaE protein encoded by the nucleotide sequence shownin SEQ ID NO:5.
 77. The feed of claim 38 wherein the hyaE gene encodes ahyaE protein in which amino acids 239-359 are deleted, wherein the aminoacids are numbered according to the hyaE protein encoded by thenucleotide sequence shown in SEQ ID NO:5.
 78. The method of claim 50wherein the hyaE gene encodes a hyaE protein in which amino acids239-359 are deleted, wherein the amino acids are numbered according tothe hyaE protein encoded by the nucleotide sequence shown in SEQ IDNO:5.